This project involves a comprehensive approach toward the design and implementation of a therapy that will allow for solid organ allotransplants to be performed with a reduced requirement for maintenance immunosuppression. Therapies are evaluated in non-human primates (NHPs) for translation into clinical trials. The primary pre-clinical model is the rhesus monkey kidney (or in some cases skin) transplant model. In vitro studies serve to define mechanisms that are potential targets for therapeutic manipulation.[unreadable] Previous study suggests that treatment with biologicals designed to interrupt costimulatory pathways at the time of primary alloantigen exposure prevent acute allograft rejection and induce durable allospecific tolerance. In particular, several rodent studies have suggested that interruption of many T-cell costimulatory pathways induces a sustained state of allograft acceptance. Lasting, donor-specific allograft acceptance has been achieved without recipient morbidity using brief perioperative treatments with antibodies specific for molecules in the CD28-B7 and CD40-CD154 pathways. If this were true in humans, costimulation-based treatments would greatly reduce the expense and morbidity of transplantation. However, rodent models are overly permissive and infrequently reflect the clinical applicability of novel therapies. Furthermore, human-specific biologics must typically be evaluated in cross-reactive primate models prior to use in humans. These studies thus test the efficacy of costimulation-specific therapies in models that determine the generalizability of rodent work, and thus support translation to humans when appropriate.[unreadable] We have built upon previous work in this model with costimulation-blockade to demonstrate that treatment with the humanized CD154 specific monoclonal antibody (Mab) hu5c8 prevents renal and skin allograft rejection and has limited utility in reversing established rejection in NHPs. We have also demonstrated that donor specific transfusion (DST) synergizes with anti-CD154 in primates as does the mTOR inhibitor sirolimus. We have therefore initiated trials in NHPs designed to optimize the timing, dose, route and source of DST. In primates, we now have evidence that portal venous infusion of unfractionated bone marrow given preoperatively in combination with sirolimus, but not tacrolimus, greatly delays rejection with no other immunosuppressive therapy. We have now shown that preoperative DST synergizes with anti-CD154 to lead to lasting allograft survival, but does not improve the results above that seen with peripheral blood infused whole blood DST. We have also demonstrated that use of alefacept in lieu of anti-CD154 extends allograft survival but not to the extent anti-CD154 does. [unreadable] One realization coming from the CD154 therapy literature is that CD154 is expressed not only on T cells, but also on activated platelets. Indeed, one drawback of anti-CD154 has been its association with thromboembolic events, perhaps related to the interaction with platelets. We have therefor initiated studies in rodents to determine whether platelet derived CD154 is relevant to allograft rejection. Using combinations of CD154 knock out mice, adoptive platelet transfusions, recombinant CD154 and cardiac transplants, we have shown that platelet derived CD154 is indeed sufficient to induce rejection without any not platelet source of this molecule. This is the first demonstration of a non-lymphocyte derived axis that mediates alloimmunity and were published this year. We are now investigating the requirement for platelet localization and the role of different splice varient forms of CD154 in mediating this effect. [unreadable] In complementary studies, we have also demonstrated that monocytes have function impact within an renal allograft infiltrate and that their activation induced cytokines specifically activate local renal tubular epithelium and induce the chemotactic stpes required for the development of allograft tubulitis. Thus, in addition to plateles, monocytes responding to general surgical trauma have been now linked with clinical rejection. [unreadable] To complement clinical studies in depletional induction we have also developed a novel polyclonal depleting antibody that has better T cell depleting ability and contains enhanced blockade of costimulation molecules while leaving molecules vital for regulatory function relatively spared. This agent has now been purified and this year we demonstrated that it successfully depletes memory T cells in primates and that it prolonges allograft survival better than clinically available formes of polyclonal antibody.[unreadable] Ischemia reperfusion injury is a known stimulator of alloimmunity. Through a collaboration with Mark Gladwin, we have provided NHP model support for the evaluation of Nitrite as a method for limiting vasospastic renal injurry and acute tubular necrosis following renal transplantation. In addition, we have initatiated studies in mice investigating a novel antagonist of annexin-V receptor function to determine if blockade of this pathway limits macrophage influx into a recently re-perfused kidney and in doing so, prevents immune mediated reperfusion injury.[unreadable] Again, in studies complementary to our clinical trials, we have identified a sub-population of cells (effector memory cells) that appear to be critically important in mediating rejection. We are now using polychromatic flow cytometry to distinguish these cells from central memory or naive cells in an attempt to determine if cells likely to induce rejection can be prospectively identified and targted for specific elimination prior to transplantation.